Acoustically mediated fluid transfer methods and uses thereof

ABSTRACT

Acoustic waves are used to transfer small amounts of fluid in a non-contact manner. Acoustic waves are propagated through a pool of a source fluid in such a manner that causes the ejection of a single micro-droplet from the surface of the pool. The droplet is ejected towards a target with sufficient force to provide for contact of the droplet with the target. Because the fluid is not contacted by any fluid transfer device such as a pipette, the opportunities for contamination are minimized. Methods may be employed to transfer fluids from an array of source sites to an array of target sites, thereby enabling the precise automation of a wide variety of procedures including screening and synthesis procedures commonly used in biotechnology.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent applicationSer. No. 09/735,709, filed on Dec. 12, 2000, the contents of which isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to non-contact fluid transfermethods, apparatus and uses thereof.

BACKGROUND

[0003] Many methods for the precision transfer and handling of fluidsare known and used in a variety of commercial and industrialapplications. The presently burgeoning industries of biotechnology andbiopharmaceuticals are particularly relevant examples of industriesrequiring ultra-pure fluid handling and transfer techniques. Not only ispurity a concern, current biotechnological screening and manufacturingmethods also require high throughput to efficiently conduct screening ofcompound libraries, synthesis of screening components, and the like.

[0004] Current fluid transfer methods require contacting the fluid witha transfer device, e.g., a pipette, a pin, or the like. Such contactmethods dramatically increase the likelihood of contamination. Manybiotechnology procedures, e.g., polymerase chain reaction (PCR), have asensitivity that results in essentially a zero tolerance forcontamination. Accordingly, a non-contact method for fluid transferwould result in a drastic reduction in opportunities for samplecontamination.

[0005] Current biotechnology screening techniques may involve manythousands of separate screening operations, with the concomitant needfor many thousands of fluid transfer operations in which small volumesof fluid are transferred from a fluid source (e.g., a multi-well platecomprising, for example, a library of test compounds) to a target (e.g.,a site where a test compound is contacted with a defined set ofcomponents). Thus, not only the source, but also the target may comprisethousands of loci that need to be accessed in a rapid,contamination-free manner.

[0006] Similarly, biotechnology synthesis methods for the generation oftools useful for conducting molecular biology research often requiremany iterations of a procedure that must be conducted withoutcontamination and with precision. For example, oligonucleotides ofvarying lengths are tools that are commonly employed in molecularbiology research applications, as, for example, probes, primers,anti-sense strands, and the like. Traditional synthesis techniquescomprise the stepwise addition of a single nucleotide at a time to agrowing oligomer strand. Contamination of the strand with an erroneouslyplaced nucleotide renders the oligonucleotide useless. Accordingly, anon-contact method for transferring nucleotides to the reaction site ofa growing oligomer would reduce the opportunity for erroneous transferof an unwanted nucleotide that might otherwise contaminate a pipette orother traditional contact-based transfer device.

[0007] Furthermore, existing fluid transfer methods are limited, and donot conveniently and reliably produce the high efficiency, high-densityarrays. Such arrays are also useful in conducting screening, synthesis,and other techniques commonly used in biotechnology.

[0008] Accordingly, there exists a need in the art for a non-contactmethod for the precision transfer of small amounts of fluid in a rapidmanner that is easily automated to meet industry needs.

SUMMARY OF THE INVENTION

[0009] In order to overcome the deficiencies of the prior art, thepresent invention provides non-contact methods for the transfer of smallamounts of fluid. Methods according to the present invention employ theuse of acoustic waves to generate micro-droplets of fluid. In themethods, acoustic waves are propagated through a pool of a source fluidto cause the ejection of at least one, e.g., a single micro-droplet,from the surface of the pool. The droplet is ejected towards a targetwith sufficient force to provide for contact of the droplet with thetarget.

[0010] The methods of the invention are easily automated in a mannerthat provides for the processing of many different sources of fluid froman array of pools of source fluid, and further provides for an array oftarget sites to receive the micro-droplets of source fluid as they areejected from the pools of source fluid. In this manner thousands ofindividual samples of source fluid can be processed and directed to thesame or two or more (e.g., a thousands or more) separate target sitesfor further reaction, detection, and the like. Thus, the presentinvention, because of its non-contact methodology, not only has greaterintrinsic reliability than is provided by presently available liquidejection on demand and continuous stream piezoelectric type pumps, butalso is compatible with a wider variety of liquid compounds, includingliquid compounds which have relatively high viscosity and liquidcompounds which contain particulate components.

[0011] The invention provides a non-contact method for transferringsmall amounts of source fluid to a target, said method comprisingpropagating an acoustic wave from an acoustic liquid deposition emitterthrough a source fluid containment structure into a pool of sourcefluid, wherein said acoustic liquid deposition emitter is in contactwith said source fluid containment structure typically through acoupling medium which is interposed between said acoustic liquiddeposition emitter and a first surface of said source fluid containmentstructure, said pool of source fluid is on a second surface of saidsource fluid containment structure that is opposite or adjacent to saidacoustic liquid deposition emitter, and said acoustic wave causescontrolled ejection of at least one droplet of said source fluid fromsaid pool to said target.

[0012] The invention also provides a non-contact method for transferringsmall amounts of a source fluid to a separate target structure, saidmethod comprising activating a piezoelectric transducer therebypropagating an acoustic wave through a coupling medium which isinterposed between said piezoelectric transducer and a first surface ofa source fluid containment structure, wherein said source fluid iscontained on a second surface of said source fluid containment structurethat is opposite said piezoelectric transducer, and said target ispositioned to receive a droplet of fluid ejected from said source fluidas a result of propagation of said acoustic wave through said sourcefluid.

[0013] The invention further provides a method for transferring smallamounts of a source fluid from a pool selected from one of a pluralityof pools of source fluid located on a first surface of a source fluidcontainment structure, to a separate target structure without physicallycontacting said source fluid, said method comprising propagating anacoustic wave through said source fluid such that a single droplet offluid is ejected from the surface of said pool of source fluid withsufficient energy to bring said droplet into contact with said target,wherein said acoustic wave is propagated from a piezoelectrictransducer, said piezoelectric transducer is in contact, opposite to, oradjacent with said source fluid containment structure via a couplingmedium interposed between said piezoelectric transducer and a secondsurface of said source fluid containment structure, said second surfaceof said source fluid containment structure is opposite said pool ofsource fluid, and said target is opposite or adjacent to said surface ofsaid pool of source fluid.

[0014] The invention also provides an apparatus for performingnon-contact transfer of small amounts of source fluid. The apparatusincludes an acoustic liquid deposition emitter and a stage wherein thestage is configured to support a source fluid containment structuresupported such that the acoustic liquid deposition emitter is inoperative contact with the source fluid containment structure when acoupling medium is interposed there between. The apparatus may include anumber of additional elements, including, for example: an acoustic wavechannel structure that is mechanically coupled to the acoustic liquiddeposition emitter (e.g., a piezoelectric transducer) to provide fortransmission of an acoustic wave from, e.g., the piezoelectrictransducer to said coupling medium; a structure for maintaining thecoupling medium in operative contact with the acoustic liquid depositionemitter; a lens for focusing said acoustic wave; controls for varyingone or more of frequency, voltage, and duration of an energy source usedto excite the acoustic liquid deposition emitter and thereby propagatean acoustic wave; a stage actuator for user-defined positioning of thestage relative to the acoustic liquid deposition emitter; a focussingactuator for user-defined positioning of said acoustic liquid depositionemitter relative to said stage; a computer for controlling the stageactuator and/or the focussing actuator; and a fluid level detector fordetecting a level of fluid in a source fluid containment structuresupported by said stage.

[0015] The invention also provides a system for performing non-contacttransfer of small amounts of a source fluid. The system includes asource fluid containment structure, a movable stage configured tosupport the source fluid containment structure, an acoustic liquiddeposition emitter in operative contact with the source fluidcontainment structure, a coupling medium interposed between thedeposition emitter and the source fluid containment structure, and acomputer in operable communication with the acoustic liquid depositionemitter for varying one or more of frequency, voltage and duration of anenergy source used to excite the acoustic liquid deposition emitter andwherein the computer is in communication with the movable stage forpositioning the source fluid such that operative contact with theacoustic liquid deposition emitter.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 is a schematic diagram illustrating one embodiment of anon-contact fluid transfer apparatus of the present invention.

[0017]FIG. 2 is a schematic diagram illustrating one embodiment of thepresent invention, where an acoustic wave 10 generated by apiezoelectric element 60 is propagated through a wave channel 70, acoupling medium 20 and a source fluid containment structure 30 to a poolof source fluid 40, causing ejection of a droplet 50 of source fluidfrom the surface of the pool.

[0018]FIG. 3 is a schematic diagram illustrating an embodiment of thepresent invention where each pool of source fluid 40 is confined by acoating of a hydrophobic material 80 on source fluid containmentstructure 30.

[0019]FIG. 4 depicts a lens contemplated for use in the practice of thepresent invention, and shows various parameters that may be adjusted toprovide correct focus of an acoustic wave. The diameter of the apertureis 2a, Z₀ is the focal length, d_(z) is the depth of field, and d_(r) isthe deposition feature diameter.

[0020]FIG. 5 is a schematic diagram illustrating several options formonitoring source fluid pool levels by monitoring acoustic wavesgenerated by secondary piezoelectric elements 65 directed at the sourcefluid pool.

[0021]FIG. 6 is a schematic diagram illustrating an embodiment of thepresent invention where a computer 100 receives signals generated by asecondary piezoelectric element 65 and computes source fluid pool 105levels from the information received. The computer of 100 then compares110 the computed height versus the emitter position 115 and controls thefocus of the primary piezoelectric element by moving the emitter (60 ofFIG. 2) relative to position the source fluid pool (40 of FIG. 2) usingpositioning stage 120 to most effectively eject droplet(s) from thesurface of the source fluid pool (40 of FIG. 2).

DETAILED DESCRIPTION OF THE INVENTION

[0022] In accordance with the present invention, there is provided anapparatus useful for non-contact fluid transfer. With reference to FIG.1 there is schematically presented one embodiment of an apparatus of thepresent invention. The figure depicts a non-contact fluid transferapparatus 5 having at least one acoustic liquid deposition emitter 60 inelectrical communication with a computer 95. During operation theacoustic liquid deposition emitter 60 generates an acoustic wave or beam10 that can be propagated through an optional wave channel 70. Theacoustic wave can be focused by tens 75 prior to propagating throughcoupling fluid 20 to optimize the energy of the acoustic wave or beam 10upon the liquid/air interface of source fluid 40. The acoustic wave 10is propagated through a coupling medium 20 after which the wave istransmitted through source fluid containment structure 30 where the wavecomes to focus at or near the surface of a pool of source fluid 40thereby causing ejection of at least one droplet 50 of source fluid fromthe surface of the pool. In one embodiment, the ejected droplet 50 makescontact with a target 80. The source fluid containment structure 30 canbe held on a movable stage 35. The movable stage 35 is controlled byactuator mechanism 85 which contains a horizontal actuator 85′ or avertical actuator 85″ or a combination of the two actuators to controlthe movement of the stage 35 in both the vertical and horizontaldirections. The actuator 85 is typically in communication with computer95 which controls the movement of the stage to select a source fluid 40or to adjust focusing of the acoustic wave or beam 10 upon the sourcefluid 40. The computer may have implemented thereon various algorithmsto adjust the focal length and energy of the acoustic deposition emitteras well as control and manage the location of the acoustic depositionemitter relative to a particular source fluid present in or on a sourcefluid containment structure.

[0023] In accordance with the present invention, there are providednon-contact methods for transferring small amounts of source fluid to atarget. The methods of the invention comprise propagating an acousticwave from an acoustic liquid deposition emitter through a source fluidcontainment structure into a pool of source fluid. The acoustic liquiddeposition emitter is coupled with the source fluid containmentstructure typically through a coupling medium, which is interposedbetween the acoustic liquid deposition emitter and a first surface ofthe source fluid containment structure. The pool of source fluid is on asecond surface of the source fluid containment structure, and the secondsurface is opposite the first surface, which is in contact with thecoupling medium. Thus, the acoustic wave is emitted from the acousticliquid deposition emitter, propagates through the coupling medium,across or through the source fluid containment structure to causecontrolled ejection of at least one droplet of the source fluid from thepool to the target. By “at least one droplet” means one or more dropletsor a plurality of droplets. The droplets can be ejected substantiallysimultaneously or sequentially. In preferred embodiments a singleindividual droplet is ejected using the methods of the invention.

[0024] For an example of one embodiment of the present invention,reference is made to FIG. 2 which shows the propagation of an acousticwave 10 through a coupling medium 20 after which the wave is transmittedthrough source fluid containment structure 30 where the wave comes tofocus at or near the surface of a pool of source fluid 40 therebycausing ejection of at least one droplet 50 of source fluid from thesurface of the pool.

[0025] As used in the context of the coupling medium, “coupled with” or“coupled to” means that the coupling medium provides a medium for theacoustic waves to travel between the acoustic liquid deposition emitterand the fluid containment structure. In a preferred embodiment, thecoupling medium is in contact with both the acoustic liquid depositionemitter and a first surface of the fluid containment structure (e.g.,the underside of the structure, if the fluid containment structure isoriented with source fluid on its top surface).

[0026] As used herein, “controlled ejection” means that the acousticwave can be adjusted, as further described herein, to vary the sizeand/or number of droplets ejected from the surface of the pool. Suchcontrolled ejection techniques can involve adjusting or focusing of theacoustic wave, frequency of the acoustic wave, modifying the distancebetween the acoustic liquid deposition emitter and the source fluid, andthe like, in response to the type of source fluid (e.g., the sourcefluids content, viscosity and the like) as well as changes in the volumeor level of the source fluid during ejection of droplets or as a resultof evaporation.

[0027] Accordingly, the methods of the invention rely on the fact thatan acoustic wave may be propagated through a pool of fluid in a mannerthat causes ejection of a single droplet of fluid from the surface ofthe pool of fluid to assist in transferring the fluid from the sourcepool to a desired target. Because the acoustic wave is of sufficientenergy and properly focused to eject at least one droplet of fluid fromthe surface of the source pool, no pipettes or other fluid handlingdevices need come into contact with the source pool of fluid.

[0028] Any type of fluid is suitable for use in the practice of thepresent invention. As used herein, “fluid” means an aggregate of matterin which the molecules are able to flow past each other without limitand without the formation of fracture planes. Thus, as recognized bythose of skill in the art, a fluid may comprise a liquid and/or a gasunder the appropriate conditions. A fluid may be homogenous, i.e., onecomponent, or heterogeneous, i.e., more than one component. Where aplurality of pools of source fluid are employed in the practice of thepresent invention, each pool may comprise a different source fluid, asfurther described herein.

[0029] “Non-contact,” as used herein, means that a source fluid istransferred or removed from a source pool of fluid without contactingthe source fluid with a transfer device. In one embodiment, the sourcefluid is transferred from a source pool of fluid to a target withoutcontacting the source fluid with a transfer device. For example, theejection of a droplet of source fluid from the source fluid containmentstructure does not contact anything other than the target towards whichthe droplet is directed. Thus, no pipettes, pins, capillaries or otherfluid transfer devices are brought into contact with the source fluid.In this manner, opportunities for contamination of the fluid and thesystem are minimized. Moreover, non-contact fluid transfer as describedherein does not require the use of nozzles with small ejection orificesthat easily clog. In addition, the relatively high cost piezoelectrictransducers and acoustic focusing lenses remain as fixed components ofthe fluid delivery/transfer system, while the fluid(s) beingtransferred, as well as the fluid containment structure, may constituteseparate and disposable components. This allows for a greatly improvedcost of ownership because the relatively high cost piezoelectrictransducer(s) and acoustic focusing lens(es) never contact theindividual fluid compounds.

[0030] As used herein, “source fluid containment structure” is anystructure suitable for containing or supporting a pool of source fluidand which allows an acoustic wave to propagate from a first side or endof the structure, through the structure to the second side or end of thestructure, wherein the source fluid is contained on the second side orwithin the structure. Thus, suitable source fluid containment structuresinclude a flat structure such as a slide (e.g., a glass or polystyrenemicroscope slide), or the like, onto which one or more discrete pools ofsource fluid may be deposited; also included are single and multi-wellplates commonly used in molecular biology applications; capillaries(e.g., capillary arrays); and the like.

[0031] Maintaining discrete pools of source fluid may be accomplished bya variety of methods, including providing a plurality of separationstructures such as wells, tubes or other devices that have at least onewall separating one fluid from another, or by providing coatings thatserve to define containment regions (as further described herein), andthe like. Immiscible fluids or materials can be used to separatedissimilar fluids (e.g., waxy coatings separating hydrophilic fluids)forming containment fields.

[0032] Source fluid containment structures may be constructed of anysuitable material, bearing in mind the need for good acoustic velocityproperties. Such materials include glass, polymer (e.g., polystyrene),metal, a textured material, a containment field, and the like, as wellas combinations thereof. The material may further be porous ornon-porous, or combinations thereof.

[0033] Source fluid containment structures may have one or more coatingsto facilitate fluid containment. Thus, in one embodiment of the presentinvention, slides having zones of relative hydrophobicity andhydrophilicity may be employed as source fluid containment structures.In this manner, an aqueous fluid may be applied to a zone of the slidethat is surrounded by a relatively hydrophobic region (or coating ofrelatively hydrophilic material), thereby operating to contain a pool ofsource fluid. Reference is made to FIG. 3 as one example of thisembodiment of the present invention. Source fluid containment structure30 has zones or regions (i.e., containment zones) for containingdiscrete pools of source fluid 40. These zones are defined by a coatingof a hydrophobic material 80 which acts to confine the source fluid 40in the containment zones. Accordingly, in this embodiment of the presentinvention, sample wells are not required to contain the discrete poolsof source fluid.

[0034] Examples of hydrophobic coatings include polytetratfluoroethylene(PTFE), hydrophobic amino acids, polypeptides comprising hydrophobicamino acids, waxes, oils, fatty acids, and the like. Those of skill inthe art can readily determine a number of other hydrophobic coatings,which may also serve to define source fluid containment zones, andcontain source fluids therein. Optionally, the zone(s) of the slidewhich are chosen to contain non-aqueous source fluid may have relativelyhydrophilic regions (or coating of relatively hydrophilic material) tofurther define the containment zone(s). Thus, pools of source fluid canbe confined to defined areas of a slide by virtue of the relative areasof hydrophobicity and hydrophilicity. Again, sample wells are notrequired to contain a pool of source fluid.

[0035] The methods of the invention are contemplated for use in highthroughput operations. It is preferred that the source fluid containmentstructure have multiple containment regions, preferably in an arraywhich can be mapped so that each containment region can be accessedunder direction of a controlling computer. Thus, in one preferredembodiment of the present invention, the source fluid containmentstructure is a multi-well plate such as a micro-titer plate (comprisinga plurality of wells, each having a bottom, sides and an open top forthe ejection of a droplet there through). Suitable micro-titer platesmay have from about 96 to about 1500 wells, or more. One example of asuitable plate is a 1536 well plate (e.g., catalog number 3950 availablefrom Corning Corporation).

[0036] As used herein, “target” means a structure or a zone towardswhich a droplet of source fluid is ejected, or with which the ejecteddroplet makes contact. The target may be constructed of any materialthat is suitable for receiving the ejected fluid droplet, including, forexample, a glass, a polymer, a paper, a gel, a conductive material, ametal, a porous material, a non-porous material, a textured material, orthe like. The material may be further coated or textured to receive andretain the droplet of fluid. Coatings contemplated for use in thepractice of the present invention include polytetrafluoroethylene(PTFE), aminomethylated or highly crosslinkedpolystyrene-divinyl-benzene, and the like. In some embodiments of thepresent invention, it may be desirable to direct a fluid droplet to ameasuring device or other remotely located zone, thus, the target maynot comprise a tangible object but instead comprise a collection zonedefined by a containment field, a conduit, a chamber, a collector, acontainer, or the like. In this manner, a droplet of fluid could bedirected, for example, to a conduit that leads to the reaction chamberof a mass spectrometer, or the like.

[0037] In one embodiment, the target is separate in that it is not incontact with the source fluid containment structure, but rather can beheld in place at a selected distance from the source containmentstructure. Of course the distance must be within the effective range ofthe droplet generated by the acoustic liquid deposition emitter. Thedroplets of the size ejected from the source pool are small (e.g., atleast about 1 micrometer), that in a vacuum they travel a relativelylarge distance (i.e., many centimeters) in opposition to the force ofgravity. One of skill in the art will recognize that the distance saidejected source material can travel will depend upon the size and contentof the ejected fluid and the surrounding atmospheric humidity,temperature and the like. In addition, the properties of the acousticwave (e.g., frequency and the like) generated by the liquid depositionemitter can be varied to adjust the distance and size of the ejectedsource fluid droplet. The formation of a given droplet is thus dependenton, for example, the frequency of the liquid deposition emitter's (e.g.,a piezoelectric transducer's) oscillation. Accordingly, a target (instill air) positioned about two (2) centimeters above the surface of thesource pool can easily be impacted with a droplet ejected from thesurface of the source pool. Thus, while a distance in excess of amillimeter can be employed in the practice of the present invention, itis presently preferred that the target be positioned no more than about0.25 millimeter from the surface of the source pool; and in a anotherpreferred embodiment, the target is no more than about five (5)millimeters from the surface of the source pool.

[0038] As used herein, “acoustic deposition emitter” means any devicecapable of generating a directional acoustic wave capable of causingejection of at least one droplet of fluid from the surface of a pool offluid. As understood by those of skill in the art, an acoustic wave orbeam exerts a radiation pressure against objects upon which it impinges.Thus, when an acoustic wave or beam impinges on a free surface (e.g.,fluid/air interface) of a pool of fluid from beneath, the radiationpressure which it exerts against the surface of the pool may reach asufficiently high level to release at least one individual droplet offluid from the pool, despite the restraining force of surface tension.In a preferred embodiment, a piezoelectric transducer is employed as anacoustic deposition emitter. In one embodiment, a piezoelectrictransducer comprises a flat thin piezoelectric element, which isconstructed between a pair of thin film electrode plates. As isunderstood by those of skill in the art, when a high frequency andappropriate magnitude voltage is applied across the thin film electrodeplates of a piezoelectric transducer, RF energy will cause thepiezoelectric element to be excited into a thickness mode oscillation.The resultant oscillation of the piezoelectric element generates aslightly diverging acoustic beam of acoustic waves. By directing thewave or beam onto an appropriate lens having a defined radius ofcurvature (e.g., a spherical lens, or the like), the acoustic beam canbe brought to focus at a desired point.

[0039] The radiation pressure is greatest in the acoustic wave or beam'sfocal region, particularly, at the pool surface where wave reflectionoccurs. The pressure caused by the acoustic wave or beam acts to lift asmall column of liquid which appears initially as a small mound. Whenenough energy is applied to overcome surface tension the mound becomes amomentary liquid fountain where each tone burst emits a single droplet.Because the focused wave or beam is diffraction limited, the dropletdiameter is proportional to the wavelength. Observations with waterindicate that single droplet ejection occurs at a specific power levelband where uniformly sized droplets form. However above this band, asone increases power level further the droplets begin to form tails whichthen break off into satellite droplets. Further increases in powercauses the process to transition to a continuous fountain.

[0040] At energy levels just below the threshold of normal dropletejection, a fine mist may be emitted from the source fluid. The mist maybe used in situations where it is desirable to coat a surface with finedroplet coating that is {fraction (1/10)} to {fraction (1/100)} the sizeof the normally produced droplets.

[0041] Fountain ejection can be achieved when the power level is wellbeyond the normal single droplet ejection range. Fountains appear to becontinuous or nearly continuous streams of liquid that eject and breakup in a random fashion and produce widely distributed sizes. This modemay be used for producing a spray like coating.

[0042] In addition, it is possible to affect the trajectory of theejected droplet by means of electrostatics. The same principals are usedin the common cathode ray tube. A simple charging plate positionedparallel to the pool surface is used. The pool acts as an opposing platesimilar to a capacitor. Therefore, the pool will acquire charge that isopposite that of the charging plate. When a droplet is ejected itcarries a isolated charge at point where it breaks off the pool. A smalldiameter hole in the charging plate permits droplet charging withoutimpeding its path. There is an acceleration experienced by the dropletso that its final velocity will be the combination of initial ejectionvelocity and an electrostatic acceleration. The charge electrode voltagemay be manipulated to accelerate droplets if higher velocity is desired.

[0043] Deflection is accomplished in a manner identical to the cathoderay tube. The deflection plates set up an electric field perpendicularto the droplets flight path. An acceleration perpendicular to the pathresults in a deflected trajectory. By manipulating the deflectionvoltage in two axes a sweep pattern is formed.

[0044] Accordingly, to eject individual droplets from the source fluidcontainment structure on demand, the RF excitation of the piezoelectricelement is amplitude or frequency modulated (by means well understood tothose of skill in the art), thereby causing the focused acoustic beamradiation pressure exerted against the surface of the source pool offluid to swing above and below a predetermined droplet ejectionthreshold level. Thus, the RF voltage applied to the piezoelectricelement may be amplitude or frequency modulated and/or energy durationmodulated to control the droplet ejection process. In a preferredembodiment, the RF excitation voltage is computer controlled and may bechanged to account for changes in the viscosity and surface tension ofthe source fluid.

[0045] In one embodiment, a computer sends an analog voltage pulse tothe piezoelectric transducer by an electrical wire. The voltage pulsecan be controlled, for example, by a MD-E-201 Drive Electronicsmanufactured by Microdrop, GmbH, Muhlenweg 143, D-22844 Norderstedt,Germany. The electronics can thus control the magnitude and duration ofthe analog voltage pulses, and also the frequency at which the pulsesare sent to the piezoelectric transducer. Each voltage pulse causes thegeneration of an acoustic wave from the piezoelectric transducer, whichin turn is propagated through a coupling medium and into or through thesource fluid thereby impinging on the surface of the source fluid. Forexample, an acoustic wave (e.g., a pressure wave) propagates through thecoupling medium and source fluid where one droplet of source fluid isemitted under high acceleration. The size of these droplets has beenshown to be very reproducible. The high acceleration of the source fluidminimizes or eliminates problems caused by source fluid surface tensionand viscosity, allowing extremely small droplets to be expelled from thesurface of a pool of source fluid, e.g., as small as 5 picoliterdroplets have been demonstrated.

[0046] The piezoelectric transducer may employ a flat crystal disk, orother crystal designs, e.g., square, perforated disk, and the like. In apresently preferred embodiment, the piezoelectric transducer is a flatdisk. Because most electronic circuits are designed for a 50 Ω (ohm)load, it is presently preferred to employ a 50 Ω (ohm) transducer. Whileany material may be used in the piezoelectric element, in a presentlypreferred embodiment of the invention, a Navy Type I piezoelectricmaterial is employed in a disk element having diameter D=0.039 inch orD=0.991 mm. Other shapes of piezoelectric crystals are also contemplatedfor use in the practice of the present invention.

[0047] Firing of the acoustic deposition emitter may be conductedmanually or under direction of a controlling computer. Because thepresent invention is useful in high throughput operations, it ispresently preferred that firing of the acoustic deposition emitter becomputer controlled. Firing of the emitter can be coordinated withcomputer controlled positioning of both the source containment structureor the target so that a specific source fluid can be directed to aspecifically selected target spot on the target.

[0048] Proper focus of the acoustic wave can be achieved by providing alens between the piezoelectric transducer and the coupling medium.Lenses contemplated for use in the practice of the present invention maybe of constant curvature or aspherical. An aspherical lens (i.e., a lenshaving a compound curvature) may be employed to accommodate anyirregularities in the acoustic wave, whether due to the piezoelectricelement itself, a misalignment of the piezoelectric element with thesurface of the pool of source fluid, or the like.

[0049] To capture the maximum amount of energy emitted by the crystal,it is preferred that the lens aperture be greater than the crystaldiameter. With reference to FIG. 4, the lens can be constructed with aspherical cutter, for example, to have a selected focal distance Z_(O).It is preferred that Z_(O)=0.125 inch or 3.175 mm. This yields anf-value (f=Z_(O)/D) equal to four (4), where D is the diameter of theactive area of the piezoelectric material. It is preferred that theradius of curvature of the lens be chosen to provide an f-value in therange of about 1 to 4. In another aspect of this embodiment, the f-valueis in the range of 1-2. In yet another aspect of this embodiment, thef-value is in the range of 2-4.

[0050] To efficiently capture the energy in the acoustic wave generatedby the piezoelectric crystal, it is desirable that the diameter of thelens be greater than the diameter of the active portion of thepiezoelectric crystal. Thus, in view of the preferred active crystaldiameter of 0.039 inches or 0.99 mm, the presently preferred value forthe radius of the lens (a) is about 0.016 inch or 0.40 mm (see FIG. 4).In a typical embodiment, the focal distance of the lens may beapproximately equal to 2.5 to 3 times the diameter of the crystal.

[0051] By virtue of having an f-value in the range of 1-4, a relativelylong focal length (d_(z)) results. Consequently, the acoustic depositionemitter is functional over a wide range of depths of source pool. Inthis manner, refocusing of the emitter is not required every time thedepth of a particular sample pool is altered by the ejection of somematerial therefrom. Nonetheless, in an alternative embodiment of thepresent invention, adjusting the focus of the acoustic beam iscontemplated. Such adjustment may be made by varying the distancebetween the acoustic deposition emitter and the surface of the pool ofsource fluid. Any methods useful for varying the distance between theacoustic deposition emitter and the surface of the pool of source fluidare contemplated for use in the practice of the present invention.Focussing may be automated and controlled by computer.

[0052] By applying a particular wavelength (λ) of the acoustic wave inthe source fluid, the depth of focus can be estimated by applying theformula dz=4.88·λ·f². The wavelength (λ) of the acoustic wave can bedetermined by those of skill in the art based on the velocity of soundthrough the chosen source fluid and the frequency of the acoustic wave.Thus, when the source fluid comprises water, the relevant equations are

V _(H2O)=1496 m/s, and λ=VH2O/frequency.

[0053] Droplet diameter (d_(r)) at a given λ and f-value can bedetermined by applying the equation d_(r)=1.02·λ·f. Similarly, aselected droplet diameter can be achieved by solving the precedingequation for λ, and employing acoustic waves of that wavelength.

[0054] By applying the forgoing equations to the preferred values forvariables (f) and (a) disclosed herein, and assuming a source fluidcomprising water, the wavelength λ=75 μm; the focal length d_(z)=3.75mm; and the droplet diameter d_(r)=245 μm.

[0055] The size of the droplet can also be adjusted by modulating one ormore of frequency, voltage, and duration of the energy source used toexcite the acoustic liquid deposition emitter (e.g., a piezoelectrictransducer). Accordingly, a wide range of user-defined droplet diameterscan be achieved by employing the methods of the invention. In oneembodiment of the present invention, the defined droplet diameter is atleast about 1 micrometer. In another embodiment of the presentinvention, the defined droplet diameter is in the range of about 1micrometer to about 10,000 micrometers. In yet another embodiment of thepresent invention, the defined droplet diameter is in the range of about500 micrometers to about 1000 micrometers. In a further embodiment ofthe present invention, the defined droplet diameter is in the range ofabout 60 micrometers to about 500 micrometers. In yet another embodimentof the present invention, the defined droplet diameter is in the rangeof about 100 micrometers to about 500 micrometers. In another embodimentof the present invention, the defined droplet diameter is in the rangeof about 120 micrometers to about 250 micrometers. In a furtherembodiment of the present invention, the defined droplet diameter is inthe range of about 30 micrometers to about 60 micrometers. In stillanother embodiment of the present invention, the defined dropletdiameter is about 50 micrometers.

[0056] It is preferred that acoustic waves be channeled from the liquiddeposition emitter (e.g., piezoelectric element) to the source fluid viaan acoustic wave channel. Reference is made to FIG. 2 which shows anacoustic wave 10 being generated by a piezoelectric element 60 andpropagated through acoustic wave channel 70. The rapid oscillation ofthe piezoelectric element 60 generates an acoustic wave 10, whichpropagates through the acoustic wave channel 70 at a relatively highvelocity until it strikes the focusing lens 75. The wave then emergesinto a medium 20 (i.e., the coupling medium) having a much loweracoustic velocity, so the spherical shape of the lens imparts aspherical wave-front to it, thereby forming the acoustic beam. Theacoustic wave channel 70 may be constructed of aluminum, silicon,silicon nitride, silicon carbide, sapphire, fused quartz, certainglasses, or the like. In a preferred embodiment, the acoustic wavechannel 70 is constructed of aluminum. Each of the aforementionedmaterials is chosen because of its high acoustic velocity typeproperties. In general, suitable materials have an acoustic velocity,which is higher than the acoustic velocity of the source fluid. It isalso preferred that the piezoelectric element 60 is deposited on orotherwise intimately mechanically coupled to a surface of the acousticwave channel 70.

[0057] In a preferred embodiment, a sufficiently high refractive indexratio is maintained between the acoustic wave channel and the sourcecontainment structure by providing a temperature controlled liquidtransition interface (e.g., a temperature controlled coupling medium asdescribed herein) that couples the highly focused acoustic wave or beamwith a containment structure. The focusing lens should direct the beaminto an essentially diffraction limited focus at or near the fluid/airinterface at the surface of the source fluid pool.

[0058] As used herein “coupling medium” means a fluid medium having anacoustic impedance that is substantially the same as the acousticimpedance of the source fluid containment structure. The coupling mediumwill be in contact with both an acoustic liquid deposition emitter orpreferably the acoustic wave channel and one side of the fluidcontainment structure, thereby providing for efficient energy transferfrom the acoustic wave channel to the fluid containment structure, andsubsequently through the source fluid. As an example, a polystyrenemulti-well plate has an acoustic impedance of about 2.3. Water has anacoustic impedance of about 1.7. Accordingly, water is a good couplingmedium when the source fluid containment structure is a polystyrenedevice (e.g., a multi-well plate) due the close match in impedencevalues between water and the plate. By adding other fluids (e.g.,glycerol, or the like) to the water, an even closer match can beachieved. Other fluids may also be employed in the practice of thepresent invention.

[0059] Thus, by providing a coupling medium between the acoustic wavedeposition emitter or preferably the acoustic wave channel and the fluidcontainment structure, a far more efficient transfer of energy occursthan if no coupling medium is employed. In one aspect of the invention,the coupling medium is temperature controlled to minimize any effect oftemperature on the source fluid.

[0060] Because these methods may be employed in high throughputapplications, it is preferred that methods of the invention furthercomprise user-defined positioning of the acoustic liquid depositionemitter relative to an array of source wells, thus providing foruser-defined association of the acoustic liquid deposition emitter witha selected pool of source fluid for ejection of a droplet therefrom.This can be accomplished by a variety of methods. For example, in thecase where a multi-well plate is employed as the source fluidcontainment structure, a computer-controlled translator (e.g., anactuator, or the like) can manipulate the position of the multi-wellplate or a movable stage upon which the multiwell plate rests. Thus, aselected well or a selected succession of wells is placed over theacoustic deposition emitter, as the source fluid contained in each wellis needed for the application being conducted (e.g., oligonucleotidesynthesis, or the like). In a related embodiment, the acousticdeposition emitter may be moved rather than the source plate. Forexample, the source fluid containment structure may remain fixed inposition and the acoustic liquid deposition emitter may be movedrelative to a well or particular source fluid of interest contained inor on the source fluid containment structure. In yet another embodiment,multiple deposition emitters may be utilized each associated, forexample, with a source fluid pool. In this embodiment, neither thesource fluid containment structure nor the deposition emitter are movedbut rather the deposition emitters are selectively activated dependingupon which source fluid is desired to have at least one droplet ejectedthere from. Once again, this allows for the selective association of theemitter with a selected source pool. Accordingly, a source fluid arrayhaving a plurality of different source fluid materials may have dropletsselectively ejected from a particular source fluid towards, for example,a target.

[0061] The target may comprise an array of target zones or target spotsto which source fluid is directed. As described above, with respect tothe source fluid and acoustic deposition emitter, the target may also bemoveable relative to a source fluid. For example, the target may bemoved relative to a source fluid to be ejected thereby allowing forselected receipt at the target of a desired ejected source fluiddroplet. The target may be positioned so that each target zone can beselectively positioned over the selected pool of source fluid. Acomputer controlled actuator arm, or the like can accomplish positioningof the target. It is presently preferred that both the target and thesource fluid containment structure be positionable via separatecomputer-controlled actuators. Thus, the non-contact fluidtransfer/deposition technology described herein provides for precisetargeting of individual source fluids to selected target zones.

[0062] Source fluids contemplated for use in the practice of the presentinvention may comprise one or more source materials. Source materialsmay include both biological and chemical compounds, agents and lifeforms (e.g., plant cells, eukaryotic or prokaryotic cells).

[0063] As used herein, “biological compounds” may comprise nucleic acids(e.g., polynucleotides), peptides and polypeptides (including antibodiesand fragments of antibodies), carbohydrates (e.g., oligosaccharides),and combinations thereof. In some embodiments, cells (e.g., eukaryoticor prokaryotic) may be contained in the fluid. Such an embodiment wouldallow for the transfer of organisms from one source fluid to anotherfluid or target during cell culturing or sorting.

[0064] The term “polynucleotides” and “oligonucleotides” include two ormore nucleotide bases (e.g., deoxyribonucleic acids or ribonucleicacids) linked by a phosphodiester bond. Accordingly, suchpolynucleotides and oligonucleotides include DNA, cDNA and RNAsequences. Polynucleotides and oligonucleotides may comprise nucleotideanalogs, substituted nucleotides, and the like. Nucleic acidscontemplated for use in the practice of the present invention includenaked DNA, naked RNA, naked plasmid DNA, either supercoiled or linear,and encapsulated DNA or RNA (e.g., in liposomes, microspheres, or thelike). As will be understood by those of skill in the art, particlesmixed with plasmid so as to “condense” the DNA molecule may also beemployed.

[0065] Polypeptides contemplated for use in the practice of the presentinvention includes two or more amino acids joined to one another bypeptide bonds. Thus, polypeptides include proteins (e.g., enzymes (e.g.,DNA polymerase), structural proteins (e.g., keratin), antibodies,fragments thereof, and the like), prions, and the like.

[0066] “Chemical compounds” contemplated for use in the practice of thepresent invention may comprise any compound that does not fall under thedefinition of biological compounds as used herein. Specific chemicalcompounds contemplated for use in the practice of the present inventionincludes dyes, detectable labels, non-enzyme chemical reagents,dilutents, and the like.

[0067] As used herein, the terms “detectable label”, “indicating group”,“indicating label” and grammatical variations thereof refer to singleatoms and molecules that are either directly or indirectly involved inthe production of a detectable signal. Any label or indicating agent canbe linked to or incorporated in a nucleic acid, a polypeptide,polypeptide fragment, antibody molecule or fragment thereof and thelike. These atoms or molecules can be used alone or in conjunction withadditional reagents. Such labels are themselves well known in the art.

[0068] The detectable label can be a fluorescent-labeling agent thatchemically binds to proteins without denaturation to form a fluorochrome(dye) that is a useful immunofluorescent tracer. Suitable fluorescentlabeling agents are fluorochromes such as fluorescein isocyanate (FIC),fluorescein isothiocyanate (FITC),5-dimethylamine-1-naphthalenesulfonylchloride (DANSC),tetramethylrhodamine isothiocyanate (TRITC), lissamine, rhodamine 8200sulphonyl chloride (RB-200-SC), and the like. A description ofimmunofluorescence analytic techniques is found in DeLuca,“Immunofluorescence Analysis”, in Antibody as a Tool, Marchalonis etal., eds., John Wiley & Sons, Ltd., pp. 189-231 (1982), which isincorporated herein by reference.

[0069] The detectable label may be an enzyme, such as horseradishperoxidase (HRP), glucose oxidase, and the like. In such cases where theprincipal indicating label is an enzyme, additional reagents arerequired for the production of a visible signal. Such additionalreagents for HRP include hydrogen peroxide and an oxidation dyeprecursor such as diaminobenzidine. An additional reagent useful withglucose oxidase is 2,2′-azino-di-(3-ethyl-benzthiazoline-G-sulfonicacid) (ABTS).

[0070] In another embodiment, radioactive elements are employed aslabeling agents. An exemplary radiolabeling agent is a radioactiveelement that produces gamma ray emissions, positron emissions, or betaemissions. Elements that emit gamma rays, such as ¹²⁴I, ¹²⁵I, ¹²⁶I, ¹³¹Iand ⁵¹Cr, represent one class of radioactive element indicating groups.Beta emitters include ³²P, ¹¹¹Indium, ³H and the like.

[0071] The linking of a label to a substrate (e.g., labeling of nucleicacids, antibodies, polypeptides, proteins, and the like), is well knownin the art. For instance, antibody molecules can be labeled by metabolicincorporation of radiolabeled amino acids provided in the culturemedium. See, for example, Galfre et al., Methods of Enzymology, 73:3-46(1981). Conventional means of protein conjugation or coupling byactivated functional groups are particularly applicable. See, forexample, Aurameas et al., Scandinavia Journal of Immunology. Vol. 8,Suppl. 7:7-23 (1978), Rodwell et al., Biotech., 3:889-894 (1984), andU.S. Pat. No. 4,493,795.

[0072] In one embodiment, the methods of the present invention may beused to pair certain ligands (i.e., a molecular group that binds toanother entity to form a larger more complex entity) and bindingpartners for such ligands. For example, certain biological molecules areknown to interact and bind to other molecules in a very specific manner.Essentially any molecules having a high binding specificity or affinityfor each other can be considered a ligand/binding partner pair, e.g., avitamin binding to a protein, a hormone binding to a cell-surfacereceptor, a drug binding to a cell-surface receptor, a glycoproteinserving to identify a particular cell to its neighbors, an antibody(e.g., IgG-class) binding to an antigenic determinant, anoligonucleotide sequence binding to its complementary fragment of RNA orDNA, and the like.

[0073] Such pairings are useful in screening techniques, synthesistechniques, and the like. Accordingly, in one embodiment of the presentinvention, screening assays may be performed in which the bindingspecificity of one compound for another is sought to be determined. Forexample, multiple test compounds (i.e., putative ligands, optionallyhaving detectable labels attached) may be screened for specificinteraction with a selected binding partner. Such assays may be carriedout by positioning one of a plurality of putative ligands in each poolof an array of source fluids. The target may comprise, for example, anarray of target zones, each zone having affixed to it a sample of thebinding partner for which specific binding is sought to be identified.Employing the methods of the invention, a droplet of each putativeligand can be ejected to a target zone and the target thereafter washedunder defined conditions. Afterwards, each of the target zones isinspected to determine whether binding of the putative ligand hasoccurred. Binding of a putative ligand serves to identify that compoundas a ligand for the binding partner. Binding can easily be identified byany method known to those of skill in the art. By employing detectablelabeled test compounds, binding can readily be determined by identifyinga labeled compound bound to the target. Of course, such assays may bereversed, i.e., the selected binding partner may be used as a labeledsource compound, while putative ligands are arrayed onto the target.

[0074] In one aspect of the foregoing embodiment, the methods of theinvention may also be applied to the identification of peptides orpeptide mimetics that bind biologically important receptors. In thisaspect, a plurality of peptides of known sequence can be applied to atarget to form an array using methods described herein. The resultingarray of peptides can then be used in binding assays with selectedreceptors (or other binding partners) to screen for peptide mimetics ofreceptor agonists and antagonists. Thus, the invention provides a methodfor producing peptide arrays on a target, and methods of using suchpeptide arrays to screen for peptide mimetics of receptor agonists andantagonists.

[0075] The specific binding properties of binding partners to ligandshave implications for many fields. For example, the strong bindingaffinity of antibodies for specific antigenic determinants is criticalto the field of immunodiagnostics. Additionally, pharmaceutical drugdiscovery, in many cases, involves discovering novel drugs havingdesirable patterns of specificity for naturally occurring receptors orother biologically important binding partners. Many other areas ofresearch exist in which the selective interaction of binding partnersfor ligands is important and are readily apparent to those skilled inthe art.

[0076] The methods of the invention may also be employed in synthesisreactions. For example, in another embodiment of the present invention,employing monomeric and/or multimeric nucleotides as source compoundscan be employed to synthesize oligonucleotides (useful as probes,labels, primers, anti-sense molecules, and the like). Such sourcecompounds may be present in a fluid medium (i.e., source fluid) and eachsource fluid placed in a defined position of an array on the sourcecontainment structure. By ejecting source nucleotides from the sourcecontainment structure onto a defined target zone of the target, definednucleotides can be added to a growing product oligonucleotide chain inan additive manner that serves to define the nucleotide sequence of thegrowing product oligonucleotide.

[0077] The particular chemical reactions necessary to performoligonucleotide synthesis are well known to those of skill in the art.Such reactions, or others, which may become known, can be performed insitu on the target by, for example, contacting the growingoligonucleotide with the necessary reagents between each iterativeaddition of further nucleotide(s). Flowing the reagents across thetarget, by passing the target through a reagent bath, or the like canperform reagent contacting. By employing a target with a suitablecoating or having suitable surface properties, the growingoligonucleotide can be bound to the target with sufficient strength toundergo the necessary chemical reactions, after which the matureoligonucleotide can be released from the target. For example, methodsfor attaching oligonucleotides to glass plates in a manner suitable foroligonucleotide synthesis are known in the art. Southern, Chem. abst.113; 152979r (1990), incorporated by reference herein in its entirety,describes a stable phosphate ester linkage for permanent attachment ofoligonucleotides to a glass surface. Mandenius et al., Anal. Biochem.157; 283 (1986), incorporated by reference herein in its entirety,teaches that the hydroxyalkyl group resembles the 5′-hydroxyl ofoligonucleotides and provides a stable anchor on which to initiate solidphase synthesis. Other such binding/release technologies are also knownor may become available and are thus contemplated for use in thepractice of the present invention.

[0078] The efficiency of oligonucleotide synthesis can be greatlyenhanced by employing nucleotide building blocks that are a combinationof monomers and multimers. Examples of nucleotide building blocksinclude nucleotides, analogues or derivatives thereof containingreactive, blocking or other groups rendering the nucleotide buildingblock suitable for reaction to form oligonucleotides. Thus, in aparticular aspect of the forgoing synthesis embodiment, there areprovided methods for oligonucleotide synthesis in which each source poolcontains an aliquot comprising one member from the group consisting ofan oligonucleotide of 10 or more nucleic acid bases, a dimericoligonucleotide (e.g., all possible combinations of an oligonucleotidecomprising two nucleotide bases), a trimeric oligonucleotide (e.g., allpossible combinations of an oligonucleotide comprising three nucleotidebases), a tetrameric oligonucleotide (e.g., all possible combinations ofan oligonucleotide comprising four nucleotide bases), and a pentamericoligonucleotide (e.g., all possible combinations of an oligonucleotidecomprising five nucleotide bases). As used herein a nucleotide base isselected from the group consisting of adenine, cytosine, guanine andthymine (or uracil). A complete set of all possible nucleotidecombinations equals 1,024 possible pentamers combinations, 256tetramers; combinations, 64 trimers combinations, 16 dimerscombinations, and 4 monomers, which can easily be placed into anindustry standard 1,536 well plate, as only 1,364 individual wells arerequired of the total 1,536 available. A computer can determine the mostefficient synthesis scheme for a desired product oligonucleotide byoptimally selecting building blocks from the source fluid wellscontaining the oligonucleotide material comprising the monomer throughpentamer oligonucleotides, and thereby minimize the number of stepsrequired to synthesize the desired product oligonucleotide. For example,the present invention allows for the synthesis of 1.0995×10¹² possible20-mer oligonucleotide combinations with only 4 couplings using anycombination of the pentamer source fluid materials. Similarly, 12couplings of any combination of the pentamer source fluid materials willgive rise to 1.329×10³⁶ possible 60-mer oligonucleotide combinations.Thus, oligonucleotide synthesis can be automated and conducted withgreater efficiency than if the synthesis were conducted by the stepwiseaddition of single nucleotides only. Other extended sequence iterativesynthesis reactions may also be performed by the methods of theinvention.

[0079] In a further embodiment of the present invention, there areprovided methods for determining or confirming the nucleotide sequenceof an “unknown” polynucleotide. The polynucleotide may be labeled byconventional methods (e.g., fluorescent, magnetic or nuclear) and thencontacted with target oligonucleotides of known sequence that havepreviously been bound to an array of sites on the target using themethods of the invention (i.e., ejection of the known oligonucleotidefrom a source pool to a desired target zone on the target array).Indeed, the target oligonucleotides may be synthesized in situ on thetarget array using methods described herein. Following contacting of the“unknown” polynucleotide with the target array of oligonucleotides, thetarget array is washed at the appropriate stringency and the presenceand location of hybridized-labeled polynucleotide is determined usingscanning analyzers or the like. Since the sequence of the targetoligonucleotide at each position of the target array is known, thisembodiment of the invention provides for the unambiguous determinationof the nucleotide sequence of the selected polynucleotide.

[0080] In performing the methods of the invention, the volume of each ofthe source pools is depleted as material is ejected from them. Thus, itis desirable to monitor the volume or level of each source pool toensure fluid is available. The volume of level of source fluid is alsoimportant because the impinging acoustic wave or beam will ejectdroplets from the surface of the source pool most efficiently if thebeam is focused as nearly as possible on the surface of the pool. Thus,by monitoring the volume or level of the source pool, the focus of theacoustic wave or beam can be adjusted (e.g., by adjusting the distancebetween the acoustic deposition emitter the source fluid containmentstructure).

[0081] Accordingly, in a further embodiment the invention provides amethod for detecting the amount of source fluid remaining in a sourcepool. Fluid volume or level detection may be performed by a variety ofmethods including direct visual/optical inspection, indirectmeasurement, and the like. In one aspect of this embodiment, detectingis performed by optically observing a change in the source fluid volumeor level as a result of ejecting said droplet from said pool. In thisaspect, optical observation may be performed by an optical detectorcoupled to a computer, wherein the computer computes a change in volumeor level based on signals received from the optical detector beforeejection of a droplet, and after the ejection of a droplet.

[0082] Optical detectors contemplated for use in the practice of thepresent invention may include a camera, a photoelectric cell, and thelike. For example, a laser or other light source can be directed at thesurface of a source pool and the defraction angle determined by one ormore photoelectric cells coupled to a computer. The angle can thusindicate the level of fluid in the source pool, and from there, thevolume can readily be computed. Other optical detection methods known tothose of skill in the art or developed in the future may also beemployed in this aspect of the present invention.

[0083] In another aspect of the invention, detection of the fluid level(volume and/or height) may be by observing the acoustic reflectionproperties of the pool of source fluid. For example, by detecting thereflection of the acoustic beam employed to eject the droplet from thesurface, the volume can be computed based on empirically determinedacoustic reflection characteristics. Since the acoustic liquiddeposition emitter (e.g., a piezoelectric transducer) design is similarwith acoustic measuring devices the droplet generator's transducer mayalso be used for acoustic depth sensing as a means of pool level orvolume feedback measurement. The signal can be processed and the systemcan then be adjusted to further focus the acoustic wave or beam as thelevel or volume changes. In another aspect of this embodiment, asecondary piezoelectric transducer can be employed to generate theacoustic beam employed to detect the fluid level. The secondarypiezoelectric transducer may be toroidal and disposed around theperimeter of the piezoelectric transducer used to eject the droplet offluid (i.e., the primary transducer). One example of this embodiment isdepicted in FIG. 5, which shows two options for deploying a secondarypiezoelectric transducer. For example, a toroidal secondary transducer65 may be disposed around the perimeter of the primary piezoelectrictransducer 60. In another aspect, a non-toroidal secondary transducer65′ may be employed to generate the acoustic wave used to gauge fluidlevel. Other deployments of the second piezoelectric transducer may alsobe employed in the practice of the present invention.

[0084] Any of the forgoing embodiments for detecting fluid level may beemployed in conjunction with a computer that retains and/or manipulatesthe values of the fluid level. In one embodiment of the presentinvention, an example of which is shown in FIG. 6, the liquid surfacereflection of the signal from the secondary piezoelectric element isreceived by a computer 100 which computes the fluid level and sends afluid level value 105 through a comparator algorithm 110 which may thenbe used to send a signal to an actuator 120 that operates to modulateone or more parameters (e.g., energy used to fire the piezoelectricelement, distance of the piezoelectric element and/or lens from thesurface of the source pool, and the like) in order to achieve thedesired focus and energy of the acoustic wave. If desired, a returnsignal 115 of one or more values such as emitter position or the like,can be returned to the comparator algorithm for further evaluation.

[0085] In addition, a computer can be used to control any number ofcontrollable parameters including, for example, a stage locationrelative to the deposition emitter (e.g., piezoelectric transducer),frequency, voltage and duration of an energy source used to excite theacoustic liquid deposition emitter.

[0086] The various techniques, methods, and aspects of the inventiondescribed above can be implemented in part or in whole usingcomputer-based systems and methods. Additionally, computer-based systemsand methods can be used to augment or enhance the functionalitydescribed above, increase the speed at which the functions can beperformed, and provide additional features and aspects as a part of orin addition to those of the invention described elsewhere in thisdocument. Various computer-based systems, methods and implementations inaccordance with the above-described technology are presented below.

[0087] A computer useful in the invention can be a processor-basedsystem including a main memory, preferably random access memory (RAM),and can also include a secondary memory. The secondary memory caninclude, for example, a hard disk drive and/or a removable storagedrive, representing a floppy disk drive, a magnetic tape drive, anoptical disk drive, etc. The removable storage drive reads from and/orwrites to a removable storage medium. Removable storage media representsa floppy disk magnetic tape, optical disk, etc., which is read by andwritten to by removable storage drive. As will be appreciated, theremovable storage media includes a computer usable storage medium havingstored therein computer software and/or data. The stored data and/orsoftware can include instructions to cause the computer to control amovable stage, frequency, voltage and duration of an energy source usedto excite the acoustic liquid deposition emitter, for example.

[0088] In alternative embodiments, secondary memory may include othersimilar means for allowing computer programs or other instructions to beloaded into a computer system. Such means can include, for example, aremovable storage unit and an interface. Examples of such can include aprogram cartridge and cartridge interface (such as the found in videogame devices), a movable memory chip (such as an EPROM, or PROM) andassociated socket, and other removable storage units and interfaceswhich allow software and data to be transferred from the removablestorage unit to the computer system.

[0089] The computer system can also include a communications interface.Communications interfaces allow software and data to be transferredbetween computer system and external devices. Examples of communicationsinterfaces can include a modem, a network interface (such as, forexample, an Ethernet card), a communications port, a PCMCIA slot andcard, etc. Software and data transferred via a communications interfaceare in the form of signals which can be electronic, electromagnetic,optical or other signals capable of being received by a communicationsinterface. These signals are provided to the communications interfacevia a channel capable of carrying signals and can be implemented using awireless medium, wire or cable, fiber optics or other communicationsmedium. Some examples of a channel can include a phone line, a cellularphone link, a RIF link, a network interface, and other communicationschannels. The computer interface or communications ports can be used toreceive instructions or to cause an apparatus operably connected to thecomputer to perform a particular function.

[0090] Without further elaboration, it is believed that one skilled inthe art can, using the preceding description, utilize the presentinvention to its fullest extent. The following examples are intended toillustrate but not to limit the invention in any manner, shape, or form,either explicitly or implicitly. While they are typical of those thatmight be used, other procedures, methodologies, or techniques known tothose skilled in the art may alternatively be used.

[0091] While the invention has been described in detail with referenceto certain preferred embodiments thereof, it will be understood thatmodifications and variations are within the spirit and scope of thatwhich is described and claimed.

That which is claimed:
 1. A non-contact method for transferring smallamounts of source fluid, said method comprising propagating an acousticwave from an acoustic liquid deposition emitter through a source fluidcontainment structure into a source fluid, wherein: a) said acousticliquid deposition emitter is in contact with said source fluidcontainment structure through a coupling medium which is interposedbetween said acoustic liquid deposition emitter and a first surface ofsaid source fluid containment structure, b) said source fluid is on asecond surface of said source fluid containment structure that isopposite said acoustic liquid deposition emitter, and c) said acousticwave causes controlled ejection of at least one droplet of said sourcefluid from said pool.
 2. A method according to claim 1, wherein said atleast one droplet contacts a target after being ejected from the surfaceof said source fluid.
 3. A method according to claim 1, wherein saidacoustic liquid deposition emitter comprises a piezoelectric transducerfor generation of said acoustic wave.
 4. A method according to claim 3,wherein said piezoelectric transducer is mechanically coupled to anacoustic wave channel structure, wherein said acoustic wave channelstructure has an acoustic impedance that is greater than the acousticimpedance of said source fluid.
 5. A method according to claim 3,wherein said acoustic liquid deposition emitter further comprises a lensfor focusing said acoustic wave.
 6. A method according to claim 5,wherein said lens is spherical.
 7. A method according to claim 5,wherein said lens has an f value in the range of about 1 to about
 4. 8.A method according to claim 1, wherein said at least one droplet has adefined diameter.
 9. A method according to claim 8, wherein said defineddiameter is in the range of about 1 micrometer to about 1000micrometers.
 10. A method according to claim 8, wherein said definedsize is controlled by varying one or more of frequency, voltage, andduration of an energy source used to excite a piezoelectric transducerand thereby propagate said acoustic wave.
 11. A method according toclaim 1, wherein said source fluid is contained within a well having abottom, sides and an open top for the ejection of said droplet therethrough.
 12. A method according to claim 1, wherein said source fluidcontainment structure comprises one or more regions of hydrophilicityfor containing said source fluid.
 13. A method according to claim 2,wherein said source fluid containment structure comprises an array ofsource fluids, and wherein said target comprises an array of targetregions for receiving said droplet.
 14. A method according to claim 13,wherein each source fluid in the array of source fluids comprises adifferent source fluid.
 15. A method according to claim 13, wherein saidarray of source fluids is contained in an array of source wells, whereineach of said wells comprises a bottom, sides and an open top for theejection of said droplet there through.
 16. A method according to claim13, wherein said target region is positioned opposite a selected sourcefluid, such that liquid ejected from the selected source fluid contactssaid target region.
 17. A method according to claim 15, wherein saidmethod further comprises positioning of said acoustic liquid depositionemitter relative to said array of source wells to provide foruser-defined association of said acoustic liquid deposition emitter witha selected source fluid for ejection of at least one droplet therefrom.18. A method according to claim 17, wherein said positioning isaccomplished by computer-controlled translation of said liquiddeposition emitter with respect to said array of source wells.
 19. Amethod according to claim 17, wherein said positioning is accomplishedby computer-controlled translation of said array of source wells withrespect to said liquid deposition emitter.
 20. A method according toclaim 1, wherein said source fluid comprises one or more sourcematerials.
 21. A method according to claim 20, wherein said sourcematerials comprises one or more biological or chemical compounds.
 22. Amethod according to claim 21, wherein said source material bears adetectable label.
 23. A method according to claim 22, wherein saiddetectable label is fluorescent or radioactive.
 24. A method accordingto claim 21, wherein said biological source material comprises a nucleicacid, a polypeptide, a eukaryotic cell, a prokaryotic cell, or acombination thereof.
 25. A method according to claim 24, wherein saidnucleic acid is DNA or RNA.
 26. A method according to claim 24, whereinsaid polypeptide is an antibody, an enzyme, or an immunogen.
 27. Amethod according to claim 21, wherein said source material is a mono- oroligonucleotide, or combination thereof.
 28. A method according to claim27, wherein said oligonucleotide comprises 2 to 10 nucleotide bases. 29.A method according to claim 27, wherein said oligonucleotide comprises 5nucleotide bases.
 30. A method according to claim 27, wherein said monoor oligonucleotides are combined on a target under conditions to form asingle product oligonucleotide.
 31. A method according to claim 30,wherein in said target is functional for binding one or more sourcematerials.
 32. A method according to claim 31, wherein said target bearsone or more target materials.
 33. A method according to claim 32,wherein said target material comprise biological or chemical compounds.34. A method according to claim 33, wherein said biological targetmaterial comprises a nucleic acid, a polypeptide, or a combinationthereof.
 35. A method according to claim 34, wherein said nucleic acidis DNA or RNA.
 36. A method according to claim 32, wherein said targetmaterial is a mono- or oligonucleotide or a combination thereof.
 37. Amethod according to claim 36, wherein said oligonucleotide comprises 2or more nucleotide bases.
 38. A method according to claim 30, whereinsaid mono- or oligonucleotide are systematically combined on said targetunder conditions to form a single product oligonucleotide.
 39. A methodaccording to claim 2, wherein said target comprises a biological orchemical target material.
 40. A method according to claim 39, whereinsaid biological target material is a polypeptide, an antibody, anenzyme, or an immunogen.
 41. A method according to claim 1, furthercomprising detecting the volume or level of source fluid present in saidfluid containment structure.
 42. A method according to claim 41, whereinsaid detecting is performed by directing an acoustic wave at said sourcefluid, receiving a reflection of said acoustic wave, and determining thelevel of said source fluid based on the characteristics of saidreflected acoustic wave.
 43. A method according to claim 41, whereinsaid volume level is performed by an acoustical detector coupled to acomputer, and wherein said computer computes a change in volume or levelbased on a signal received from the detector before said ejecting ofsaid droplet, and after said ejecting of said droplet.
 44. A non-contactmethod for transferring small amounts of a source fluid to a separatetarget structure, said method comprising activating a piezoelectrictransducer thereby propagating an acoustic wave through a couplingmedium which is interposed between said piezoelectric transducer and afirst surface of a source fluid containment structure, wherein a) saidsource fluid is contained on a second surface of said source fluidcontainment structure that is opposite said piezoelectric transducer, b)said target is positioned to receive a droplet of fluid ejected fromsaid source fluid as a result of propagation of said acoustic wavethrough said source fluid.
 45. A method for transferring small amountsof a source fluid from a pool selected from one of a plurality of poolsof source fluid located on a first surface of a source fluid containmentstructure, to a separate target structure without physically contactingsaid source fluid, said method comprising propagating an acoustic wavethrough said source fluid such that a single droplet of fluid is ejectedfrom the surface of said pool with sufficient energy to bring saiddroplet into contact with said target, wherein a) said acoustic wave ispropagated from a piezoelectric transducer, b) said piezoelectrictransducer is in contact with said source fluid containment structurevia a coupling medium interposed between said piezoelectric transducerand a second surface of said source fluid containment structure, c) saidsecond surface of said source fluid containment structure is oppositesaid pool, and d) said target is opposite said surface of said pool. 46.An apparatus for performing non-contact transfer of small amounts ofsource fluid, said apparatus comprising an acoustic liquid depositionemitter and a stage wherein: a) said stage supports a source fluidcontainment structure, b) said source fluid containment structure beingsupported such that said acoustic liquid deposition emitter is inoperative contact with said source fluid containment structure when acoupling medium is interposed there between.
 47. The apparatus of claim46, wherein said apparatus further comprises an acoustic wave channelstructure that is mechanically coupled to the acoustic liquid depositionemitter.
 48. The apparatus of claim 46, wherein acoustic liquiddeposition emitter further comprises a lens for focusing an acousticwave.
 49. The apparatus of claim 48, wherein said lens is spherical. 50.The apparatus of claim 48, wherein said lens has an f value in the rangeof about 1 to about
 4. 51. The apparatus of claim 46, wherein saidacoustic liquid deposition emitter comprises a piezoelectric transducer.52. The apparatus of claim 46, further comprising controls for varyingone or more of frequency, voltage, and duration of an energy source usedto excite said piezoelectric transducer and thereby propagate anacoustic wave therefrom.
 53. The apparatus of claim 46, furthercomprising a fluid level detector for detecting a level or volume offluid in a source fluid containment structure.
 54. The apparatus ofclaim 46, wherein the stage is movable relative to the acoustic liquiddeposition emitter.
 55. The apparatus of claim 46, wherein the stage ismovable relative to a target.
 56. An apparatus for performingnon-contact transfer of small amounts of source fluid to a target, saidapparatus comprising an acoustic liquid deposition emitter and a stagewherein: a) said stage is configured to support a source fluidcontainment structure whereby the acoustic liquid deposition emitter iscoupled to a first surface of the source fluid containment structure bya coupling medium interposed between said acoustic liquid depositionemitter and said first surface of the source fluid containmentstructure, such that b) an acoustic wave generated by said acousticliquid deposition emitter and transmitted through said coupling mediumto said first surface of the source fluid containment structure andthereafter propagates through said source fluid containment structureinto a pool of source fluid on a second surface of said source fluidcontainment structure opposite said acoustic liquid deposition emitter,causing controlled ejection of at least one droplet of said source fluidfrom said pool.
 57. A system for performing non-contact transfer ofsmall amounts of source fluid, comprising: a source fluid containmentstructure; a movable stage, configured to support the source fluidcontainment structure; an acoustic liquid deposition emitter inoperative contact with the source fluid containment structure; acoupling medium interposed between the deposition emitter and the sourcefluid containment structure; a fluid level detector for detecting alevel or volume of fluid in a source fluid containment structure; a lensin operative association with the acoustic liquid deposition emitter forfocusing an acoustic wave; and a computer in operable communication withthe acoustic liquid deposition emitter for varying one or more offrequency of the acoustic wave; voltage of an energy source used toexcite the acoustic liquid deposition emitter; duration of an energysource used to excite the acoustic liquid deposition emitter, orlocation of the stage relative to the acoustic deposition emitter; andwherein the computer comprises a computer implemented algorithm foradjusting one or more of: frequency of the acoustic wave; voltage of anenergy source used to excite the acoustic liquid deposition emitter;duration of an energy source used to excite the acoustic liquiddeposition emitter, or location of the stage relative to the acousticdeposition emitter in response to a change in fluid level or volumedetected by the fluid level detector.